Head and neck squamous cell carcinomas (HNSCC) continue to cause significant morbidity and mortality, resulting in approximately 10,000 deaths each year in the United States. Despite more advanced treatments, survival statistics have not significantly improved in decades. Research on the mechanisms regulating growth inhibition and death in cancer cell lines has revealed that ceramide is a key regulatory molecule of these pathways, especially in HNSCC tumors. Moreover, studies have shown that ceramide regulates autophagy as either a protective mechanism or a lethal pathway in cancer cells. However, it is not understood how ceramide regulates whether autophagy will be lethal or protective for the cell. Interestingly, in a study of 45 HNSCC patients ceramide synthase-1 (CerS1) and its anti-proliferative product C (18:0)-ceramide (C18-Cer) was the only ceramide species significantly down-regulated in tumors compared to normal tissues. Additionally, low levels of C18-ceramide were associated with more extensive lymphovascular invasion as well as nodal metastasis. Published studies from our lab indicate that reconstituting biologically relevant C18-ceramide levels results in growth inhibition of HNSCC tumor growth in a murine model. Preliminary data presented in this proposal indicates that C18-ceramide suppresses HNSCC cell growth and increases death via an autophagic pathway. The long-term goal of this research proposal is to elucidate the mechanism by which C18-ceramide mediates lethal autophagy in HNSCC cells. In order to determine whether C18-ceramide induces lethal autophagy in HNSCC cells, I will use various pharmacological and molecular inhibitors of autophagy to measure the affect on cell death. Given the link between autophagy induction and ceramide induced cell death, I will use siRNA, molecular techniques, and live-cell imaging to elucidate the mechanism involved. Lastly, I will use an orthotopic HNSCC murine model to test the efficacy of a novel C18-ceramide analogue at inducing lethal autophagy thus suppressing tumor growth in vivo. Our specific aims in this proposal are: 1) Examine the role of C18-ceramide in inducing lethal or protective autophagy in situ; 2) Elucidate the mechanism by which C18-ceramide induces lethal autophagy; 3) Determine the role of C18-ceramide targeted to the tumor cells mitochondria in suppressing HNSCC tumor growth and metastasis in vivo.